Fluorescence
Fluorescence is a cyclical process where a luminescence is generated by certain molecules in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength.
In the fluorescence process, certain molecules are capable of being excited, via absorption of light energy, to a higher energy state, also called an excited state. The energy of this short-lived excited state decays (or decreases) resulting in the emission of light energy. The emission of light via this process is to “fluoresce.”
A fluorophore is a molecule that is capable of fluorescing. In its ground state, the fluorophore molecule is in a relatively low-energy, stable configuration, and it does not fluoresce. When light from an external source hits a fluorophore molecule, the molecule can absorb the light energy. If the energy absorbed is sufficient, the molecule reaches an excited state (high energy); this process is known as excitation. There are multiple excited states or energy levels that the fluorophore can attain, depending on the wavelength and energy of the external light source. Since the fluorophore is unstable at high-energy configurations, it eventually adopts the lowest-energy excited state, which is semi-stable. The excited lifetime (the length of time that the fluorophore is an excited state) is very short; the fluorophore rearranges from the semi-stable excited state back to the ground state, and part of the excess energy may be released and emitted as light. The emitted light is of lower energy, and of longer wavelength, than the absorbed light, thus the color of the light that is emitted is different from the color of the light that has been absorbed. De-excitation returns the fluorophore to its ground state. The fluorophore can absorb light energy again and go through the excited state to ground state process repeatedly.
Fluorescence Spectra
A fluorescent dye absorbs light over a range of wavelengths and every dye has a characteristic excitation range. This range of excitation wavelengths is referred to as the fluorescence excitation spectrum and reflects the range of possible excited states that the dye can achieve. Certain wavelengths within this range are more effective for excitation than other wavelengths. A fluorophore is excited most efficiently by light of a particular wavelength. This wavelength is the excitation maximum for the fluorophore. Less efficient excitation can occur at wavelengths near the excitation maximum; however, the intensity of the emitted fluorescence is reduced. Although illumination at the excitation maximum of the fluorophore produces the greatest fluorescence output, illumination at lower or higher wavelengths affects only the intensity of the emitted light; the range and overall shape of the emission profile are unchanged.
Fluorophore molecules, when excited, emit over a range of wavelengths. This range of wavelengths is referred to as the fluorescence emission spectrum. There is a spectrum of energy changes associated with these emission events. A molecule may emit at a different wavelength with each excitation event because of changes that can occur during the excited lifetime, but each emission will be within the fluorescence emission spectrum. Although the fluorophore molecules all emit the same intensity of light, the wavelengths, and therefore the colors of the emitted light, are not homogeneous. The emission maximum is the wavelength where the population of molecules fluoresces most intensely. The excited fluorophore can also emit light at wavelengths near the emission maximum. However, this light will be less intense.
The emission maximum for a given fluorophore is always at a longer wavelength (lower energy) than the excitation maximum. This difference between the excitation and emission maxima is called the Stokes shift. The magnitude of the Stokes shift is determined by the electronic structure of the fluorophore, and is characteristic of the fluorophore molecule. The Stokes shift is due to the fact that some of the energy of the excited fluorophore is lost through molecular vibrations that occur during the brief lifetime of the molecule's excited state. This energy is dissipated as heat to surrounding solvent molecules as they collide with the excited fluorophore.
Filters and Light Sources
Fluorescence requires a source of excitation energy. There are many light source options for fluorescence. Selecting the appropriate light source, and filters for both excitation and emission, can increase the sensitivity of signal detection.
Several types of light sources are used to excite fluorescent dyes. The most common sources used are broadband sources, such as, for example, mercury-arc and tungsten-halogen lamps. These lamps produce white light that has peaks of varying intensity across the spectrum. When using broadband white light sources it is necessary to filter the desired wavelengths needed for excitation; this is most often done using optical filters. Optical filters selectively allow light of certain wavelengths to pass while blocking out undesirable wavelengths. A bandpass excitation filter transmits a narrow range of wavelengths and may be used for selective excitation.
Laser excitation sources provide wavelength peaks that are well-defined, selective, and of high intensity allowing more selective illumination of the sample. The best performance is achieved when the dye's peak excitation wavelength is close to the wavelength of the laser. Several lasers commonly used include, for example, the compact violet 405 nm laser, 488 nm blue-green argon-ion laser, 543 nm helium-neon green laser, and 633 nm helium-neon red laser. Mixed-gas lasers such as, for example, the krypton-argon laser, can output multiple laser lines which may require optical filters to achieve selective excitation. High-output light-emitting diodes (LEDs) provide selective wavelengths, low cost and energy consumption, and long lifetime. Single-color LEDs are ideal for low-cost instrumentation where they can be combined with simple longpass filters that block the LED excitation and allows the transmission of the dye signal. However, the range of wavelengths emitted from each LED is still relatively broad and also may require the use of a filter to narrow the bandwidth.
Filters are important for selecting excitation wavelengths and for isolating the fluorescence emission emanating from the dye of interest. Stray light arising from sources other than the emitting fluorophores (for example, from the excitation source) interferes with the detection of the fluorescence emission. Stray light therefore must be contained to ensure only the fluorescence of the sample registers with the instrument's light-sensitive detectors. When a single dye is used, a long pass emission filter which selectively blocks out the excitation light to reduce background noise may be used to maximize the signal collected. If multiple dyes are used in the sample, a band pass emission filter can be used to isolate the emission from each dye.
Flow Cytometry
Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multi-parametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus.
Flow cytometry utilizes a beam of light (usually laser light) of a single wavelength that is directed onto a hydro-dynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a lower frequency than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector (usually one for each fluorescent emission peak) it then is possible to derive various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).
Flow Cytometers
Flow cytometers are able to provide real-time analysis of several thousand particles every second and can actively separate and isolate particles having specified properties. Single-cell suspensions first must be prepared to analyze solid tissues.
A flow cytometer has five main components: 1) a flow cell where a liquid stream (sheath fluid) carries and aligns the cells so that they pass single file through the light beam for sensing; 2) a light source, such as lamps (mercury, xenon); high power water-cooled lasers (argon, krypton, dye laser); low power air-cooled lasers (argon (488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV)); or diode lasers (blue, green, red, violet); 3) a detector and Analogue to Digital Conversion (ADC) system for generating FSC and SSC as well as fluorescence signals; 4) an amplification system (linear or logarithmic): and 5) a computer for analysis of the signals.
Early flow cytometers were generally experimental devices, but recent technological advances have created a considerable market for the instrumentation, the reagents used, such as, for example, fluorescently-labeled antibodies, and analysis software. Modern instruments usually have multiple lasers and fluorescence detectors; up to 4 lasers and 18 fluorescence detectors within a single instrument are available. Increasing the number of lasers and detectors allows for multiple antibody labeling, and can identify a target population by its phenotype. Certain instruments can take digital images of individual cells more precisely, allowing for the analysis of fluorescent signal location within or on the surface of cells.
The use of fluorescent molecules, such as fluorophore-labeled antibodies, in flow cytometry is a common way to study cellular characteristics. Within these types of experiments, a labeled antibody is added to the cell sample. The antibody then binds to a specific molecule on the cell surface or inside the cell. Finally, when the laser light of the appropriate wavelength strikes the fluorophore, a fluorescent signal is emitted and detected by the flow cytometer.
The data generated by flow cytometers can be plotted in a single dimension, to produce a histogram, or in two dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions (referred to as “gates”). Specific gating protocols exist for diagnostic and clinical purposes especially in relation to hematology. The plots often are made on logarithmic scales. Signals at the detectors have to be compensated electronically as well as computationally due to emission spectra overlap of different fluorophores. Data accumulated using the flow cytometer may be exported to be re-analyzed elsewhere, freeing up the instrument for other researchers to use.
FACS
Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. The term “FACS” is not a generic term for flow cytometry, although many immunologists inappropriately use the term FACS for all types of sorting and non-sorting applications.
Utilizing FACS, a cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet. Before the stream breaks into droplets the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring or plane is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the prior light scatter and fluorescence intensity measurements, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems the charge is applied directly to the stream while a nearby plane or ring is held at ground potential and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off.
Fluorescence Detection
For proper data interpretation, the fluorescent light recorded from one fluorescent source must be distinguished from that recorded from other fluorescent sources. For that reason, the ideal fluorophore has a fluorescence emission profile of a very intense, narrow peak that is well separated from all other emission peaks. Typical organic dyes and fluorescent proteins, however, have broad emission peaks that may overlap, (i.e., emit some light in the same wavelength range). This spectral overlap may compromise data and analysis.
Multiple Fluorescent Signals
Background fluorescence, which may originate from endogenous sample constituents (autofluorescence) or from unbound or nonspecifically bound reagents, may severely compromise fluorescence detection. FIG. 2 shows fluorescence detection of mixed species. Excitation (EX) in overlapping absorption bands A1 and A2 produces two fluorescent species with spectra E1 and E2. The detection of autofluorescence (represented here by the A2-E2 spectra) can be minimized either by selecting filters that reduce the transmission of E2 relative to E1 or by selecting reagents that absorb and emit at longer wavelengths. Although narrowing the fluorescence detection bandwidth increases the resolution of E1 and E2, it also compromises the overall fluorescence intensity detected. Signal distortion caused by autofluorescence of cells, tissues and biological fluids is most readily minimized by using reagents that can be excited at >500 nm. At longer wavelengths, light scattering by dense media such as tissues is much reduced, resulting in greater penetration of the excitation light. The use of optical filters isolate quantitative emission signals S1 and S2.
Multicolor labeling incorporates the use of two or more probes to simultaneously monitor different biochemical functions. This technique has major applications in flow cytometry, DNA sequencing, fluorescence in situ hybridization and fluorescence microscopy. Signal isolation and data analysis are facilitated by maximizing the spectral separation of the multiple emissions (E1 and E2 in FIG. 2). Consequently, fluorophores with narrow spectral bandwidths, such as, for example, Alexa Fluor dyes and BODIPY dyes (Molecular Probes, Eugene, Oreg.), are useful in multicolor applications. An ideal combination of dyes for multicolor labeling would exhibit strong absorption at a coincident excitation wavelength and well-separated emission spectra (FIG. 2). Unfortunately, it is not simple to find single dyes with the requisite combination of a large extinction coefficient for absorption and a large Stokes shift.
Signal Amplification
Fluorescence signals may be amplified by increasing the number of fluorophores available for detection. However, simply increasing the probe concentration can be counterproductive and often produces marked changes in the probe's chemical and optical characteristics. The effective intracellular concentration of probes loaded by bulk permeabilization methods usually is much higher (>10-fold) than the extracellular incubation concentration. Additionally, the increased labeling of proteins or membranes ultimately leads to precipitation of the protein or gross changes in membrane permeability. Antibodies labeled with more than four to six fluorophores per protein may exhibit reduced specificity and reduced binding affinity. At high degrees of substitution, the extra fluorescence obtained per added fluorophore typically decreases due to self-quenching.
Compensation
The presence of multiple fluorescent signals must be accommodated within any fluorescence detection system for accurate quantification and analysis. Fluorescence is recorded using an emission filter chosen to collect the maximum amount of light coming from the fluorophore of interest and to exclude as much light as possible from other nearby fluorophores or fluorescent sources. While an emission filter efficiently captures the emission peak of the target fluorophore, it also may collect the light from one or more additional fluorophores due to spectral overlap in the emission profiles. The term “compensation” as used herein refers to correction of the emission signal to accurately estimate the fluorescence signal for a given fluorophore. A percentage of fluorescence is subtracted from one channel measuring a fluorophore from a second channel measuring the fluorescence of the second (or multiple) fluorophore such that the contribution of the incidental fluorescence is removed. Depending upon the instrument and software used, compensation may be set either in the instrument hardware before the sample is run or within the software after data collection. Every fluorophore combination that shows spectral overlap must be compensated. To determine the amount of compensation required to correct the fluorescence data, single-color samples (either aliquots of the cell sample stained with each fluorophore separately or microspheres that capture an individual reagent) are utilized and analyzed in parallel with the experimental samples stained with multiple fluorophores.
The complex methodologies involved in fluorescence and fluorescence detection provide significant hurdles for the researcher to consider. The further complexities of flow cytometry, combined with the consequent design of experimental protocol and detailed analysis involving numerous fluorophores and fluorescent signals, provide additional obstacles for the efficient utilization of flow cytometry. Proper consideration of spectral overlap that results from use or inclusion of multiple fluorescent materials in different detection systems currently is reactive to these problems as they arise.
There is a need in the art for identifying, important dye-detector spectral overlaps for use in designing flow cytometry experiments.